Genome Editing Compositions and Methods for Treatment of Amyotrophic Lateral Sclerosis

Abstract

Provided herein are prime editing methods and compositions for treatment of genetic disorders such as amyotrophic lateral sclerosis.

What is claimed is: 
     
         1 . A composition comprising a first prime editing guide RNA (PEgRNA) and a second PEgRNA, wherein:
 the first PEgRNA comprises a first spacer that is complementary to a first search target sequence on a first strand of a double-stranded C9ORF72 gene, a first gRNA core that associates with a first prime editor comprising a DNA binding domain and DNA polymerase domain, and a first editing template; and   the second PEgRNA comprises a second spacer that is complementary to a second search target sequence on a second strand of the double-stranded C9ORF72 gene, a second gRNA core that associates with a second prime editor comprising a DNA binding domain and a DNA polymerase domain, and a second editing template,   wherein the first strand and the second strand of the double-stranded C9ORF72 gene are complementary to each other, and wherein the first editing template and the second editing template each comprises a region of complementarity to each other.   
     
     
         2 . A composition comprising a first prime editing guide RNA (PEgRNA) and a second PEgRNA, wherein:
 the first PEgRNA comprises a first spacer that is complementary to a first search target sequence on a first strand of a double-stranded C9ORF72 gene, a first gRNA core that associates with a first prime editor comprising a DNA binding domain and DNA polymerase domain, and a first editing template; and   the second PEgRNA comprises a second spacer that is complementary to a second search target sequence on a second strand of the double-stranded C9ORF72 gene, a second gRNA core that associates with a second prime editor comprising a DNA binding domain and a DNA polymerase domain, and a second editing template,   wherein the first strand and the second strand of the double-stranded C9ORF72 gene are complementary to each other, wherein the first editing template comprises a region of identity to a sequence on the first strand of the C9ORF72 gene, and wherein the second editing template comprises a region of identity to a sequence on the second strand of the double-stranded C9ORF72 gene.   
     
     
         3 . The composition of  claim 1 or 2 , wherein the first PEgRNA directs the first prime editor to generate a first nick on the second strand of the C9ORF72 gene, wherein the second PEgRNA directs the second prime editor to generate a second nick on the first strand of the C9ORF72 gene, and wherein the C9ORF72 gene comprises an inter-nick duplex (IND) between the position of the first nick and the position of the second nick. 
     
     
         4 . The composition of  claim 3 , wherein the IND comprises an array of hexanucleotide repeats. 
     
     
         5 . The composition of  any one of the preceding claims , wherein the double-stranded C9ORF72 gene comprises a mutation associated with amyotrophic lateral sclerosis. 
     
     
         6 . The composition of  claim 5 , wherein the IND comprises the mutation associated with amyotrophic lateral sclerosis. 
     
     
         7 . The composition of  claim 6 , wherein the mutation is an increased number of hexanucleotide repeats in the array of hexanucleotide repeats compared to a wild-type C9ORF72 gene. 
     
     
         8 . The composition of any one of  claims 4-7 , wherein the array of hexanucleotide repeats comprises the sequence (GGGGCC) n  or the complementary sequence (GGCCCC) n  thereof, wherein n is any integer greater than 22. 
     
     
         9 . The composition of  claim 8 , wherein n is an integer greater than 49. 
     
     
         10 . The composition of  claim 9 , wherein n is an integer greater than 99. 
     
     
         11 . The composition of any one of  claim 1 or 3-10 , wherein the first editing template comprises an exogenous sequence compared to the C9ORF72 gene. 
     
     
         12 . The composition of any one of  claim 1 or 3-10 , wherein the second editing template comprises an exogenous sequence compared to the C9ORF72 gene. 
     
     
         13 . The composition of  claim 11 or 12 , wherein the region of complementarity between the first editing template and the second editing template comprises an exogenous sequence compared to the C9ORF72 gene. 
     
     
         14 . The composition of any one of  claims 11-13 , wherein the exogenous sequence comprises a marker, an expression tag, a barcode, or a regulatory sequence. 
     
     
         15 . The composition of any one of  claim 1 or 3-10 , wherein the first editing template comprises a region of complementarity to the IND on the second strand of the C9ORF72 gene. 
     
     
         16 . The composition of any one of  claim 1 or 3-10 , wherein the second editing template comprises a region of complementarity to the IND on the first strand of the C9ORF72 gene. 
     
     
         17 . The composition of  claim 15 or 16 , wherein the sequence of the region of complementarity between the first editing template and the second editing template is at least partially identical to a sequence in the IND. 
     
     
         18 . The composition of any one of  claims 15-17 , wherein the first editing template comprises the sequence (GGGGCC) n , wherein n is any integer between 0 and 22. 
     
     
         19 . The composition of  claim 18 , wherein n is any integer between 0 and 20. 
     
     
         20 . The composition of  claim 19 , wherein n is any integer between 3 and 10. 
     
     
         21 . The composition of any one of  claims 15-17 , wherein the second editing template comprises the sequence (GGCCCC) m , wherein m is any integer between 0 and 22. 
     
     
         22 . The composition of  claim 21 , wherein m is any integer between 0 and 20. 
     
     
         23 . The composition of  claim 22 , wherein m is any integer between 3 and 10. 
     
     
         24 . The composition of  claim 17 , wherein the region of complementarity between the first editing template and the second editing template comprises the sequence (GGGGCC) w , wherein w is any integer between 0 and 22. 
     
     
         25 . The composition of  claim 24 , wherein w is any integer between 0 and 20. 
     
     
         26 . The composition of  claim 24 , wherein w is any integer between 3 and 10. 
     
     
         27 . The composition of any one of  claims 18-26 , wherein (n+m−w) is an integer no greater than 22. 
     
     
         28 . The composition of  any one of the preceding claims , wherein the IND further comprises a sequence upstream of the array of hexanucleotide repeats. 
     
     
         29 . The composition of  claim 28 , wherein the sequence upstream of the hexanucleotide repeat sequence is at least 10 base pairs in length. 
     
     
         30 . The composition of  claim 29 , wherein the sequence upstream of the hexanucleotide repeat sequence is 5 to 25 base pairs in length. 
     
     
         31 . The composition of  claim 29 , wherein the sequence upstream of the hexanucleotide repeat sequence is 20 to 50 base pairs in length. 
     
     
         32 . The composition of  claim 29 , wherein the sequence upstream of the hexanucleotide repeat sequence is 50 to 100 base pairs in length. 
     
     
         33 . The composition of  claim 29 , wherein the sequence upstream of the hexanucleotide repeat sequence is 100, 200, 300, 400, or 500 base pairs in length. 
     
     
         34 . The composition of any one of  claims 28-33 , wherein the IND further comprises a sequence downstream of the array of hexanucleotide repeats. 
     
     
         35 . The composition of  claim 34 , wherein the sequence downstream of the hexanucleotide repeat sequence is at least 10 base pairs in length. 
     
     
         36 . The composition of  claim 34 , wherein the sequence downstream of the hexanucleotide repeat sequence is 5 to 25 base pairs in length. 
     
     
         37 . The composition of  claim 34 , wherein the sequence downstream of the hexanucleotide repeat sequence is 20 to 50 base pairs in length. 
     
     
         38 . The composition of  claim 34 , wherein the sequence downstream of the hexanucleotide repeat sequence is 50 to 100 base pairs in length. 
     
     
         39 . The composition of  claim 34 , wherein the sequence downstream of the hexanucleotide repeat sequence is 100, 200, 300, 400, or 500 base pairs in length. 
     
     
         40 . The composition of any one of  claims 15-39 , wherein the first editing template further comprises a region of complementarity to the sequence of the IND upstream of the array of hexanucleotide repeats. 
     
     
         41 . The composition of  claim 40 , wherein the region of complementarity of the first editing template to the sequence of the IND upstream of the array of hexanucleotide repeats is 5 to 25 nucleotides in length. 
     
     
         42 . The composition of  claim 40 , wherein the region of complementarity of the first editing template to the sequence of the IND upstream of the array of hexanucleotide repeats is 10 to 15 nucleotides in length. 
     
     
         43 . The composition of  claim 40 , wherein the region of complementarity of the first editing template to the sequence of the IND upstream of the array of hexanucleotide repeats is 20 to 50 nucleotides in length. 
     
     
         44 . The composition of  claim 40 , wherein the region of complementarity of the first editing template to the sequence of the IND upstream of the array of hexanucleotide repeats is 50 to 100 nucleotides in length. 
     
     
         45 . The composition of  claim 1 , wherein the first editing template comprises at its 5′ end, a sequence selected from the group consisting of: nucleotides 1-100 of SEQ ID NO: 110; nucleotides 1-90 of SEQ ID NO: 111; nucleotides 1-80 of SEQ ID NO: 112; nucleotides 1-70 of SEQ ID NO: 113; nucleotides 1-60 of SEQ ID NO: 114; nucleotides 1-50 of SEQ ID NO: 115; nucleotides 1-40 of SEQ ID NO: 116; 30 nucleotides 1-300f SEQ ID NO: 117; nucleotides 1-20 of SEQ ID NO: 118, nucleotides 1-10 of SEQ ID NO: 119, and SEQ ID NOs: 100-109; and 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, and 454. 
     
     
         46 . The composition of  claim 1 , wherein the first editing template comprises at its 3′ end, a sequence selected from the group consisting of: the last 100 nucleotides of SEQ ID NO: 110; the last 90 nucleotides of SEQ ID NO: 111; the last 80 nucleotides of SEQ ID NO: 112; the last 70 nucleotides of SEQ ID NO: 113; the last 60 nucleotides of SEQ ID NO: 114; the last 50 nucleotides of SEQ ID NO: 115; the last 40 nucleotides of SEQ ID NO: 116; the last 30 nucleotides of SEQ ID NO: 117; the last 20 nucleotides of SEQ ID NO: 118; the last 10 nucleotides of SEQ ID NO: 119, and SEQ ID NOs: 100-109; and 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, and 454. 
     
     
         47 . The composition of any one of  claims 45 and 46 , wherein the first editing template comprises nucleotides 1 to x of SEQ ID NO: a, wherein x is an integer from 10 to i, wherein i is the length of SEQ ID NO: a; wherein the second editing template comprises nucleotides 1 to y of SEQ ID NO: b, wherein y is an integer from (i+10−x) to i; wherein a is an integer from 100 to 119 or the integer 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, and wherein b is an integer that equals (a+99). 
     
     
         48 . The composition of any one of  claims 15-47 , wherein the second editing template further comprises a region of complementarity to the sequence of the IND downstream of the array of hexanucleotide repeats. 
     
     
         49 . The composition of  claim 48 , wherein the region of complementarity of the second editing template to the sequence of the IND downstream of the array of hexanucleotide repeats is 5 to 25 nucleotides in length. 
     
     
         50 . The composition of  claim 49 , wherein the region of complementarity of the second editing template to the sequence of the IND downstream of the array of hexanucleotide repeats is 10 to 15 nucleotides in length. 
     
     
         51 . The composition of  claim 48 , wherein the region of complementarity of the second editing template to the sequence of the IND downstream of the array of hexanucleotide repeats is 20 to 50 nucleotides in length. 
     
     
         52 . The composition of  claim 48 , wherein the region of complementarity of the second editing template to the sequence of the IND downstream of the array of hexanucleotide repeats is 50 to 100 nucleotides in length. 
     
     
         53 . The composition of  claim 1 , wherein the second editing template comprises at its 5′ end, a sequence selected from the group consisting of: nucleotides 1-100 of SEQ ID NO: 120; nucleotides 1-90 of SEQ ID NO: 121; nucleotides 1-80 of SEQ ID NO: 122; nucleotides 1-70 of SEQ ID NO: 123; nucleotides 1-60 of SEQ ID NO: 124; nucleotides 1-50 of SEQ ID NO: 125; nucleotides 1-40 of SEQ ID NO: 126; nucleotides 1-30 of SEQ ID NO: 127; nucleotides 1-20 of SEQ ID NO: 128, nucleotides 1-10 of SEQ ID NO: 129, and SEQ ID NOs: 130-139; and 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, and 455: 
     
     
         54 . The composition of  claim 1 , wherein the second editing template comprises at its 3′ end, a sequence selected from the group consisting of: the last 100 nucleotides of SEQ ID NO: 120; the last 90 nucleotides of SEQ ID NO: 121; the last 80 nucleotides of SEQ ID NO: 122; the last 70 nucleotides of SEQ ID NO: 123; the last 60 nucleotides of SEQ ID NO: 124; the last 50 nucleotides of SEQ ID NO: 125; the last 40 nucleotides of SEQ ID NO: 126; the last 30 nucleotides of SEQ ID NO: 127; the last 20 nucleotides of SEQ ID NO: 128, the last 10 nucleotides of SEQ ID NO: 129, and SEQ ID NOs: 130-139; and 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, and 455 
     
     
         55 . The composition of  claim 53 or 54 , wherein the second editing template comprises nucleotides 1 to x of SEQ ID NO: a, wherein x is an integer from 10 to i, wherein i is the length of SEQ ID NO: a; wherein the first editing template comprises nucleotides 1 to y of SEQ ID NO: b, wherein y is an integer from (i+10−x) to i; wherein a is an integer from 120 to 139 or the integer 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, or 455, and wherein b is an integer that equals (a+99). 
     
     
         56 . The composition of  claim 2-10 , wherein the first editing template and the second editing template are not complementary to each other. 
     
     
         57 . The composition of  claim 2-10 , wherein the first editing template and the second editing template comprise a region of complementarity to each other. 
     
     
         58 . The composition of  claim 57 , wherein the region of complementarity between the first editing template and the second editing template comprises an exogenous sequence compared to the double-stranded C9ORF72 gene. 
     
     
         59 . The composition of  claim 58 , wherein the exogenous sequence comprises a marker, an expression tag, a barcode, or a regulatory sequence. 
     
     
         60 . The composition of any one of  claim 1 or 3-10 , wherein the first editing template comprises a region of identity or substantial identity to a sequence on the second strand of the double-stranded C9ORF72 gene immediately adjacent to and outside the IND. 
     
     
         61 . The composition of any one of  claim 1 or 3-10 , wherein the region of identity or substantial identity of the first editing template to the sequence on the first strand of the double-stranded C9ORF72 gene immediately adjacent to and outside the IND is at least 10 nucleotides in length. 
     
     
         62 . The composition of  claim 61 , wherein the region of identity or substantial identity of the first editing template to the sequence on the first strand of the double-stranded C9ORF72 gene immediately adjacent to and outside the IND is 15 to 100 nucleotides in length. 
     
     
         63 . The composition of any one of  claim 1 or 3-10 , wherein the second editing template comprises a region of identity or substantial identity to a sequence on the second strand of the C9ORF72 gene immediately adjacent to and outside the IND. 
     
     
         64 . The composition of  claim 63 , wherein the region of identity or substantial identity of the second editing template to the sequence on the second strand of the double-stranded C9ORF72 gene immediately adjacent to and outside the IND is at least 10 nucleotides in length. 
     
     
         65 . The composition of  claim 64 , wherein the region of identity or substantial identity of the second editing template to the sequence on the second strand of the double-stranded C9ORF72 gene immediately adjacent to and outside the IND is 15 to 100 nucleotides in length. 
     
     
         66 . The composition of  any one of the preceding claims , wherein the first PEgRNA comprises a first primer binding site (PBS) sequence that comprises a region of complementarity to the second strand of the double-stranded C9ORF72 gene. 
     
     
         67 . The composition of  claim 66 , wherein the composition further comprises a second PEgRNA comprising a second PBS sequence that comprises a region of complementarity to the first strand of the double-stranded C9ORF72 gene. 
     
     
         68 . The composition of  claim 66 or 67 , wherein the first PEgRNA comprises a structure: 5′-[first spacer]-[first gRNA core]-[first editing template]-[first primer binding site sequence]-3′. 
     
     
         69 . The composition of  claim 66 or 67 , wherein the first PEgRNA comprises a structure: 5′-[first editing template]-[first primer binding site sequence]-[first spacer]-[first gRNA core]-3′. 
     
     
         70 . The composition of any one of  claims 67-69 , wherein the second PEgRNA comprises a structure: 5′-[second spacer sequence]-[second gRNA core]-[second editing template]-[second primer binding site]-3′. 
     
     
         71 . The composition of any one of  claims 67-69 , wherein the second PEgRNA comprises a structure: 5′-[second editing template]-[second primer binding site sequence]-[second spacer]-[second gRNA core]-3′. 
     
     
         72 . The composition of  claim 66 , wherein the first PBS is at least partially complementary to the first spacer sequence. 
     
     
         73 . The composition of  claim 67 , wherein the second PBS is at least partially complementary to the second spacer sequence. 
     
     
         74 . The composition of  any one of the preceding claims , wherein the first search target sequence is at least 10, 50, 100, 200, 300, 400 or 500 nucleotides upstream of the IND. 
     
     
         75 . The composition of  claim 74 , wherein the first search target sequence is at least 100 nucleotides upstream of the IND. 
     
     
         76 . The composition of  claim 74 , wherein the first search target sequence is at least 50 nucleotides upstream of the IND. 
     
     
         77 . The composition of  any one of the preceding claims , wherein the second search target sequence is at least 10, 50, 100, 200, 300, 400 or 500 nucleotides downstream of the IND. 
     
     
         78 . The composition of  claim 77 , wherein the second search target sequence is at least 100 nucleotides downstream of the IND. 
     
     
         79 . The composition of  claim 77 , wherein the second search target sequence is at least 50 nucleotides downstream of the IND. 
     
     
         80 . The composition of  claim 66 , wherein the first PBS is about 2 to 20 nucleotides in length. 
     
     
         81 . The composition of  claim 67 , wherein the second PBS is about 2 to 20 nucleotides in length. 
     
     
         82 . The composition of  claim 80 , wherein the first PBS is about 8 to 16 nucleotides in length. 
     
     
         83 . The composition of  claim 81 , wherein the second PBS is about 8 to 16 nucleotides in length. 
     
     
         84 . The composition of anyone of  claim 1 or 3-10 , wherein the first editing template is about 15-150 nucleotides in length. 
     
     
         85 . The composition of  claim 84 , wherein the first editing template is about 15-100 nucleotides in length. 
     
     
         86 . The composition of  claim 85 , wherein the first editing template is about 30-100 nucleotides in length. 
     
     
         87 . The composition of  claim 86 , wherein the first editing template is about 50-100 nucleotides in length. 
     
     
         88 . The composition of  claim 87 , wherein the first editing template is about 15-50 nucleotides in length. 
     
     
         89 . The composition of anyone of  claim 1 or 3-10 , wherein the second editing template is about 15-150 nucleotides in length. 
     
     
         90 . The composition of  claim 89 , wherein the second editing template is about 15-100 nucleotides in length. 
     
     
         91 . The composition of  claim 90 , wherein the second editing template is about 30-100 nucleotides in length. 
     
     
         92 . The composition of  claim 91 , wherein the second editing template is about 50-100 nucleotides in length. 
     
     
         93 . The composition of  claim 92 , wherein the second editing template is about 15-50 nucleotides in length. 
     
     
         94 . The composition of  any one of the preceding claims , wherein the first editing template and second editing template are of the same length. 
     
     
         95 . The composition of any one of  claims 1-93 , wherein the first editing template and second editing template are of different lengths. 
     
     
         96 . The composition of  any one of the preceding claims , wherein the first and/or the second spacer is about 12 to 20 nucleotides in length. 
     
     
         97 . The composition of  claim 96 , wherein the first and/or the second spacer is about 15 to 20 nucleotides in length. 
     
     
         98 . The composition of  claim 97 , wherein the first and/or the second spacer is about 17 to 20 nucleotides in length. 
     
     
         99 . The composition of  claim 96 , wherein the first spacer comprises a sequence selected from the group consisting of SEQ ID NOs: 34-39. 
     
     
         100 . The composition of  claim 96 , wherein the second spacer comprises a sequence selected from the group consisting of SEQ ID NOs: 40-51, 70-84, and 280-292. 
     
     
         101 . The composition of  claim 66 , wherein the first PBS comprises a sequence selected from the group consisting of SEQ ID NOs: 52-69. 
     
     
         102 . The composition of  claim 67 , wherein the second PBS comprises a sequence selected from the group consisting of SEQ ID NOs: 85-99 and 140-279. 
     
     
         103 . The composition of  claim 47 , wherein the first editing template comprises a sequence selected from the group consisting of SEQ ID NOs: 100-119 and 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454. 
     
     
         104 . The composition of  claim 55 , wherein the second editing template comprises a sequence selected from the group consisting of SEQ ID NOs: 120-139 and 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, or 455. 
     
     
         105 . A composition comprising a first prime editing guide RNA (PEgRNA) and a second PEgRNA, wherein the first PEgRNA comprises a first spacer comprising a sequence selected from the group consisting of SEQ ID NOs: 34-39, a guide RNA core comprising a sequence selected from the group consisting of SEQ ID NOs: 456-465, a first PBS comprising a sequence selected from the group consisting of SEQ ID NOs: 52-69, and a first editing template comprising a sequence selected from the group consisting of SEQ ID NOs: 100-119 and 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, and wherein the second PEgRNA comprises a second spacer comprising a sequence selected from the group consisting of SEQ ID NOs: 40-51, 70-84, and 280-292, a guide RNA core comprising a sequence selected from the group consisting of SEQ ID NOs: 456-465, a second PBS comprising a sequence selected from the group consisting of SEQ ID NOs: 85-99 and 140-279, and a second editing template comprising a sequence selected from the group consisting of SEQ ID NOs: 120-139 and 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, or 455. 
     
     
         106 . A composition of  claim 105 , wherein the PEgRNA editing pair is selected from the group comprising SEQ ID NOs: 3 and 32, SEQ ID NOs: 3 and 21, SEQ ID NOs: 2 and 21, SEQ ID NOs: 1 and 32, SEQ ID NOs: 1 and 20, SEQ ID NOs: 5 and 19, SEQ ID NOs: 16 and 22, SEQ ID NOs: 16 and 20, or SEQ ID NOs: 16 and 19. 
     
     
         107 . A composition of  claim 105 , wherein the PEgRNA editing pair is selected from the group comprising SEQ ID NOs: 3 and 33, SEQ ID NOs: 3 and 31, SEQ ID NOs: 3 and 30, SEQ ID NOs: 3 and 23, SEQ ID NOs: 3 and 20, SEQ ID NOs: 2 and 33, SEQ ID NOs: 2 and 32, SEQ ID NOs: 2 and 31, SEQ ID NOs: 2 and 29, SEQ ID NOs: 2 and 24, SEQ ID NOs: 2 and 19, SEQ ID NOs: 1 and 33, SEQ ID NOs: 1 and 31, SEQ ID NOs: 1 and 30, SEQ ID NOs: 1 and 23, SEQ ID NOs: 1 and 22, SEQ ID NOs: 1 and 21, SEQ ID NOs: 1 and 19, SEQ ID NOs: 6 and 33, SEQ ID NOs: 6 and 32, SEQ ID NOs: 6 and 31, SEQ ID NOs: 6 and 30, SEQ ID NOs: 6 and 29, SEQ ID NOs: 6 and 28, SEQ ID NOs: 6 and 20, SEQ ID NOs: 6 and 19, SEQ ID NOs: 5 and 33, SEQ ID NOs: 5 and 32, SEQ ID NOs: 5 and 31, SEQ ID NOs: 5 and 30, SEQ ID NOs: 5 and 25, SEQ ID NOs: 5 and 21, SEQ ID NOs: 5 and 20, SEQ ID NOs: 4 and 33, SEQ ID NOs: 4 and 32, SEQ ID NOs: 4 and 30, SEQ ID NOs: 4 and 21, SEQ ID NOs: 4 and 20, SEQ ID NOs: 4 and 19, SEQ ID NOs: 15 and 33, SEQ ID NOs: 15 and 30, SEQ ID NOs: 15 and 29, SEQ ID NOs: 15 and 27, SEQ ID NOs: 18 and 24, SEQ ID NOs: 18 and 22, SEQ ID NOs: 17 and 24, SEQ ID NOs: 17 and 23, SEQ ID NOs: 17 and 22, SEQ ID NOs: 16 and 24, SEQ ID NOs: 16 and 23, SEQ ID NOs: 18 and 27, SEQ ID NOs: 17 and 27, SEQ ID NOs: 16 and 25, SEQ ID NOs: 17 and 30, SEQ ID NOs: 18 and 33, SEQ ID NOs: 18 and 32, SEQ ID NOs: 18 and 31, SEQ ID NOs: 17 and 33, SEQ ID NOs: 17 and 32, SEQ ID NOs: 17 and 31, SEQ ID NOs: 16 and 33, SEQ ID NOs: 16 and 31, SEQ ID NOs: 18 and 21, SEQ ID NOs:18 and 20, SEQ ID NOs: 17 and 20, SEQ ID NOs: 17 and 19, or SEQ ID NOs: 16 and 21. 
     
     
         108 . A dual prime editing system comprising the composition of  any one of the preceding claims  and further comprising a first prime editor that comprises a DNA binding domain and a DNA polymerase domain and associates with the first PEgRNA, and a second prime editor that comprises a DNA binding domain and a DNA polymerase domain and associates with the second PEgRNA. 
     
     
         109 . The dual prime editing system of  claim 108 , wherein the first prime editor and the second prime editor are the same. 
     
     
         110 . The dual prime editing system of  claim 109 , wherein the DNA binding domain is a CRISPR associated (Cas) protein domain. 
     
     
         111 . The dual prime editing system of  claim 110 , wherein the Cas protein domain has a nickase activity. 
     
     
         112 . The dual prime editing system of  claim 111 , wherein the Cas protein domain is a Cas9. 
     
     
         113 . The dual prime editing system of  claim 112 , wherein the Cas9 comprises a mutation in an HNH domain. 
     
     
         114 . The dual prime editing system of  claim 113 , wherein the Cas9 comprises a H840A mutation in the HNH domain. 
     
     
         115 . The dual prime editing system of  claim 111  wherein the Cas protein domain is a Cas12b. 
     
     
         116 . The dual prime editing system of  claim 111 , wherein the Cas protein domain is a Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or a Casp. 
     
     
         117 . The dual prime editing system of any one of  claims 108-116 , wherein the DNA polymerase domain is a reverse transcriptase. 
     
     
         118 . The dual prime editing system of  claim 117 , wherein the reverse transcriptase is a retrovirus reverse transcriptase. 
     
     
         119 . The dual prime editing system of  claim 118 , wherein the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) reverse transcriptase. 
     
     
         120 . The dual prime editing system of any one of  claims 108-119 , wherein the DNA polymerase domain and the DNA binding domain are fused or linked to form a fusion protein, and wherein the DNA binding domain is a programmable DNA binding domain. 
     
     
         121 . A lipid nanoparticle (LNP) or ribonucleoprotein (RNP) comprising the dual prime editing system of any one of  claims 108-120 , or a component thereof. 
     
     
         122 . A polynucleotide encoding the first PEgRNA and second PEgRNA of any one of  claims 1-107 , the dual prime editing system of any one of  claims 108-121 , or a component thereof. 
     
     
         123 . The polynucleotide of  claim 122 , wherein the polynucleotide is mRNA. 
     
     
         124 . The polynucleotide of  claim 122 or 123 , wherein the polynucleotide is operably linked to a regulatory element. 
     
     
         125 . The polynucleotide of  claim 124 , wherein the regulatory element is an inducible regulatory element. 
     
     
         126 . A vector comprising the polynucleotide of any one of  claims 122-125 . 
     
     
         127 . The vector of  claim 126 , wherein the vector is an AAV vector. 
     
     
         128 . An isolated cell comprising the first PEgRNA and second PEgRNA of any one of  claims 1-103 , the dual prime editing system of any one of  claims 108-120 , the LNP or RNP of  claim 121 , the polynucleotide of any one of  claims 122-125 , or the vector of  claim 126 or 127 . 
     
     
         129 . The cell of  claim 128 , wherein the cell is a human cell. 
     
     
         130 . The cell of  claim 128 or 129 , wherein the cell is a neuron, a motor neuron cell, an upper motor neuron cell, a lower motor neuron cell, a glial cell, a myeloid cell, a lymphocyte, a T lymphocyte, a microglial cell, or a dendritic cell. 
     
     
         131 . A pharmaceutical composition comprising (i) the composition of any one of  claims 1-103 , the dual prime editing system of any one of  claims 108-120 , the LNP or RNP of  claim 121 , the polynucleotide of any one of  claims 122-125 , or the vector of  claim 126 or 127 , or the cell of any one of  claim 129 or 130 ; and (ii) a pharmaceutically acceptable carrier. 
     
     
         132 . A method for editing a C9ORF72 gene, the method comprising contacting the C9ORF72 gene with (i) the composition of any one of  claims 1-107 , (ii) a first prime editor comprising a DNA binding domain and a DNA polymerase domain that associates with the first PEgRNA, and (iii) a second prime editor comprising a DNA binding domain and a DNA polymerase domain that associates with the second PEgRNA, wherein the first PEgRNA directs the first prime editor to generate a first nick on the second strand of the C9ORF72 gene, wherein the second PEgRNA directs the second prime editor to generate a second nick on the first strand of the C9ORF72 gene, and wherein the contacting results in excision of an inter-nick duplex (IND) between the position of the first nick and the position of the second nick of the C9ORF72 gene, thereby editing the C9ORF72 gene. 
     
     
         133 . A method for editing a C9ORF72 gene, the method comprising contacting the C9ORF72 gene with the dual prime editing system of any one of  claims 108-120 ,
 wherein the first PEgRNA directs the first prime editor to generate a first nick on the second strand of the C9ORF72 gene, wherein the second PEgRNA directs the second prime editor to generate a second nick on the first strand of the C9ORF72 gene, and   wherein the contacting results in excision of an inter-nick duplex (IND) between the position of the first nick and the position of the second nick of the C9ORF72 gene, thereby editing the C9ORF72 gene.   
     
     
         134 . The method of  claim 132 or 133 , wherein the first prime editor and the second prime editor are the same. 
     
     
         135 . The method of any one of  claims 132-134 , wherein the first editing template encodes a first single-stranded DNA, and wherein the first single-stranded DNA is incorporated in the C9ORF72 gene. 
     
     
         136 . The method of any one of  claims 132-135 , wherein the second editing template encodes a second single-stranded DNA, and wherein the second single-stranded DNA is incorporated in the C9ORF72 gene. 
     
     
         137 . The method of any one of  claims 132-136 , wherein the contacting results in deletion of the array of hexanucleotide repeats in the C9ORF72 gene. 
     
     
         138 . The method of any one of  claims 132-136 , wherein the contacting results in a reduced number of hexanucleotide repeats in the C9ORF72 gene. 
     
     
         139 . The method of any one of  claims 132-136 , wherein the contacting results in deletion of the sequence (GGGGCC)X in the C9ORF72 gene, wherein x is an integer no less than 1. 
     
     
         140 . The method of  claim 139 , wherein x is an integer between 5 and 30. 
     
     
         141 . The method of  claim 139 , wherein x is an integer greater than 50. 
     
     
         142 . The method of  claim 139 , wherein x is an integer greater than 100. 
     
     
         143 . The method of  claim 139 , wherein x is an integer greater than 1000. 
     
     
         144 . The method of any one of  claims 132-143 , wherein the contacting results in no greater than 22 GGGGCC repeats in intron 1 of the C9ORF72 gene. 
     
     
         145 . The method of  claim 144 , wherein the contacting results in no GGGGCC repeats in intron 1 of the C9ORF72 gene. 
     
     
         146 . The method of any one of  claims 132-145 , wherein the C9ORF72 gene is in a cell. 
     
     
         147 . The method of  claim 146 , wherein the cell is a mammalian cell, human cell, primary cell, glial cell, or a neuron. 
     
     
         148 . The method of  claim 146 or 147 , wherein the cell is a neuron cell, a neuron progenitor cell, glial cell, or a differentiated neuron. 
     
     
         149 . The method of any one of  claims 146-148 , wherein the cell is in a subject. 
     
     
         150 . The method of  claim 149 , wherein the subject is a human. 
     
     
         151 . The method of any one of  claims 146-150 , wherein the cell is from a subject having amyotrophic lateral sclerosis. 
     
     
         152 . The method of  claim 151 , further comprising administering the cell to the subject after the contacting. 
     
     
         153 . A method for treating amyotrophic lateral sclerosis in a subject in need thereof, the method comprising administering to the subject the composition of any one of  claims 1-103 , the dual prime editing system of any one of  claims 108-120 , the LNP or RNP of  claim 121 , the polynucleotide of any one of  claims 122-125 , the vector of  claim 126 or 127 , or the cell of any one of  claims 124-126 , or the pharmaceutical composition of  claim 127 , wherein the administration results in a reduced number of an array of GGGGCC repeats in the C9ORF72 gene in the subject, thereby treating amyotrophic lateral sclerosis in the subject. 
     
     
         154 . The method of  claim 153 , wherein the subject is a human.